(1) Field of the Invention
This invention relates generally to nucleic acid analysis and, more particularly, to methods for detecting nucleic acid target sites with fluorescence labeled oligonucleotides and to use of the methods in DNA genotyping.
(2) Description of the Related Art
Nucleic acid analysis has become increasingly important in a number of applications including the genotyping of individuals such as in the diagnosis of hereditary diseases, the detecting of infectious agents, tissue typing for histocompatability, the identifying of individuals in forensic and paternity testing and monitoring the genetic make up of plants and animals in agricultural breeding programs (see, for example, Alford and Caskey, Cur Opin Biotech 5:29-33, 1994 which is incorporated by reference).
One approach to nucleic acid analysis uses probes which are complementary to a nucleotide or nucleotides in the nucleic acid. These analyses are typically performed in conjunction with amplification of the DNA being tested by the polymerase chain reaction (Saiki et al., Science 239:487-491, 1988 which is incorporated by reference). Two variations of this approach are the Genetic bit analysis method and the oligonucleotide ligation assay.
The Genetic bit analysis method involves hybridization of an oligonucleotide to a DNA sequence immediately adjacent to a target nucleotide. The oligonucleotide then undergoes a 3' extension with a labeled dideoxynucleoside triphosphate and the labeled oligonucleotide is subsequently detected using enzyme linked colorimetry. (Nikiforov et al, Nucleic Acids Res 22:4167-4175, 1994 which is incorporated by reference).
The oligonucleotide ligation assay involves hybridization of a DNA sequence to two probes, one of which is labeled. One of the probes hybridizes to the nucleotides immediately contiguous to a target nucleotide and a second, allele-specific probe hybridizes to the target nucleotide and immediately contiguous nucleotides on the opposite side to the first probe. The two probes are then ligated and the resultant labeled oligonucleotide is detected using enzyme linked colorimetry (Nickerson et al., Proc Natl Acad Sci 87:8923-8927, 1990; U.S. Pat. Nos. 4,883,750, 4,988,617 and 5,242,794 all of which are incorporated by reference).
Both the genetic bit analysis and oligonucleotide ligation assay are time consuming and not readily adaptable to automation because they require capturing, separation, and washing of the labeled oligonucleotide followed by a multi-step detection procedure using an enzyme-linked immunosorbent assay.
In another approach, the detection of one or more nucleotides in a nucleic acid is accomplished using oligonucleotide probes labeled with two fluorescent substances in close proximity. One of the fluorophores (donor) has an emission spectrum that overlaps the excitation spectrum of the other fluorophore (acceptor) and transfer of energy takes place from the donor to the acceptor through fluorescence resonance energy transfer (T. Foster, Modern Quantum Chemistry, Istanbul Lectures, Part III, 93-137, 1965, Academic Press, New York which is incorporated by reference). The energy transfer is mediated by dipole-dipole interaction. Spectroscopically, when the donor is excited, its specific emission intensity decreases while the acceptor's specific emission intensity increases, resulting in fluorescence enhancement.
The fluorescence enhancement has been used in detection systems in which either two singly labeled oligonucleotides (Heller et al., EPO patent applications 0070685, 1983 which is incorporated by reference) or one doubly labeled oligonucleotide probe (Heller, EPO patent application 0229943, 1986 which is incorporated by reference) are first prepared and then hybridized to a target DNA or RNA sequence. The two fluorescent labels are separated by less than 22 nucleotides in the case of two singly labeled oligonucleotides or from 2 to 7 intervening base units in the case of doubly labeled oligonucleotide probes such that enhanced emission from fluorescent energy transfer can be detected from the hybridized probes.
The so-called Taqman assay uses a fluorescent energy transfer detection method which takes advantage of the decrease in emission intensity, i.e. or quenching observed in the first fluorophore. (Livak et al., PCR Methods and Applications 4:357-362, 1995; U.S. Pat. No. 5,528,848 which are incorporated by reference). An oligonucleotide containing the two fluorescent substances is hybridized to a target DNA sequence. The fluorescent substances are covalently linked to the oligonucleotide at a distance such that fluorescent energy transfer takes place which is then measured as a quenching of donor fluorescence. During amplification by polymerase chain reaction, the oligonucleotide is degraded thus separating the two fluorescent substances. As a result, the donor shows a loss of quenching and increase in fluorescent emission. Thus, by monitoring the loss of quenching of the donor, the target DNA sequence is detected.
One application of the TaqMan assay is in detecting single nucleotide polymorphisms, i.e. single base mutations in DNA. This method provides significant advantages over earlier assays for single nucleotide polymorphisms which were labor intensive and not readily automated. (see, for example, Botstein et al., Am J Human Genetics 32:314-331, 1980; Hayashi, PCT Methods and Applications 1:34-38, 1991; Meyers et al., Methods in Enzymology 155:501-527, 1987; Keen et al., Trends in Genetics 7:5, 1991; Cotton et al., Proc Natl Acad Sci 85:4397-4401; Myers et al., Science 230:1242-1246, 1985; and Kwok et al., Genomics 23:138-144, 1994 which are incorporated by reference). Nevertheless, a significant problem with the TaqMan assay results from a relative intolerance to mismatches which is disadvantageous for allelic discrimination. (Livak et al, PCR Methods and Applications 4:357-362, 1995 which is incorporated by reference). Thus, there remains a continuing need for an effective nucleic acid assay method that is simple to perform and readily automated.